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(Hypertension. 1997;30:1047-1053.)
© 1997 American Heart Association, Inc.

Articles

Prostacyclin Release by Rat Cardiac Fibroblasts

Inhibition of Collagen Expression

Hisahiro Yu; Ann Marie Gallagher; Phillip M. Garfin; Morton P. Printz

From the Department of Pharmacology, University of California at San Diego, La Jolla.

Correspondence to Morton P. Printz, Department of Pharmacology 0636, University of California at San Diego, Room 3092, Basic Sciences Bldg, La Jolla, CA 92093-0636. E-mail mprintz{at}ucsd.edu

	Abstract

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Abstract Cardiac fibroblasts, as the source of extracellular matrix for the left ventricle, subserve important functions to cardiac remodeling and fibrotic development following myocardial infarction or with pressure-overload cardiac hypertrophy. The fibroblast may be the target cell for angiotensin-converting enzyme inhibitors (ACEI) that are cardioprotective and reverse collagen deposition and remodeling but whose mechanisms of action remain controversial. Because we previously documented phenotypic differences between cardiac fibroblasts from the spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) left ventricle, the present study evaluated whether phenotypic differences also exist in the release of endogenous arachidonic acid metabolites or in the activation of phospholipase D, and the importance of observed differences to the formation of collagen and the mechanism of action of ACEI. The experimental design compared endogenous sources of arachidonic acid with exogenous prelabeling of cells. Angiotensin II stimulated greater arachidonic acid release than bradykinin, and WKY cells were more responsive than SHR. The major prostanoid formed by cardiac fibroblasts was prostaglandin I₂ (PGI₂), with more prostacyclin production by WKY cells than SHR cells both under nonstimulated conditions and in response to angiotensin II or bradykinin. Beraprost, a PGI₂ analogue, was shown to decrease growth rate and DNA synthesis of fibroblasts and to inhibit mRNA expression for collagen types I and III, with SHR cells being less responsive to beraprost than WKY cells. These results potentially implicate eicosanoid metabolism, particularly PGI₂, in collagen formation, fibrotic development, and cardiac remodeling, and they imply that the SHR genetic hypertension model may be predisposed to excess cardiac fibrosis.

Key Words: hypertrophy, left ventricular • collagen types I and III • angiotensin II • arachidonic acid • bradykinin • prostacyclin

	Introduction

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Left ventricular dysfunction may result from hypertrophic and/or fibrotic processes accompanying either long-standing chronic hypertension or ventricular remodeling following acute injury such as myocardial infarction.^{1 2 3} Progressive cardiac fibrosis arises from an excess deposition and rearrangement of the ECM, predominantly fibrillar collagen types I and III, which are synthesized and secreted mainly by cardiac fibroblasts.^{4 5} However, molecular mechanisms most important to the regulation of cardiac fibroblast collagen biosynthesis and secretion remain controversial. Fibroblasts are well recognized as exhibiting plasticity, and cultured cells have been reported to retain phenotypic changes for many passages after exposure to activated monocyte secretions.⁶ We have documented stable pleiotropic differences between cultures of LV cardiac fibroblasts derived from SHR or normotensive WKY rats and from comparisons of rat, rabbit, and human cultured fibroblasts.^{7 8} These differences included variation in cell growth rate, altered Ang II receptor density and angiotensinogen mRNA expression, hormonal responsiveness, and more recently, differential peptide receptor activation of calcium signaling.

Insight into mechanisms regulating fibroblast ECM synthesis may derive from an understanding of the mechanism of action of those therapies that limit fibrosis and ventricular remodeling. ACEI are cardioprotective to both the hypertrophied and the infarcted myocardium,^{9 10 11 12} but their cardioprotective mechanisms of action remain controversial; they are attributed either to local blockade of the formation of Ang II or to the degradation of BK. Additionally, it has been suggested recently that the cardioprotective effects of ACEI are attributable to the release of nitric oxide and/or eicosanoids.^{13 14 15 16 17 18}

AA metabolites, including prostanoids such as PGE₂ and PGI₂, are known to subserve important roles on cell function, including proliferation, differentiation, and protein synthesis,^{19 20 21} and have been implicated in modulating collagen synthesis and/or deposition/accumulation.^{22 23} Additionally, strain differences in eicosanoid production by smooth muscle²⁴ or mesangial cells²⁵ derived from SHR and WKY have been reported, and altered PGE₂ formation has been linked with stable phenotypes of various noncardiac fibroblast cultures.⁶ There has been one report of eicosanoid production by cultured rat cardiac fibroblasts from exogenous AA,²⁶ but no strain comparisons were conducted. Additionally, release of AA may accompany PLD activation, and enhanced PLD activity has been reported for SHR vascular smooth muscle cells^{27 28} and has been postulated as being involved in abnormal growth by SHR cells.

The goals of the present study were first to evaluate whether strain differences exist in the release of endogenous AA metabolites or in PLD activity by cardiac fibroblasts derived from SHR and WKY left ventricles. Second, we wished to evaluate whether observed differences could be relevant to the formation of ECM and the mechanism of action of ACEI. As documented below, marked strain differences in AA metabolism stimulated by Ang II or BK were observed and provide the first evidence that a major eicosanoid formed by rat cardiac fibroblasts is PGI₂, as represented by 6-keto PGF₁, its stable metabolite. Additionally, we show that administration of a stable PGI₂ analogue exhibits inhibitory effects on cell proliferation and on gene expression for collagen types I and III.

	Methods

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Materials
[5,6,8,9,11,12,14,15-³H]AA (184.6 Ci/mmol), 6-[5,8,9,11,12,14,15-³H]keto PGF₁ (150 Ci/mmol), [9,10-³H]myristic acid (14 Ci/mmol), [methyl-³H]thymidine (83.9 Ci/mmol), and [-³²P]dCTP (3000 Ci/mmol) were from DuPont NEN; ANG II and BK were from Bachem California; indomethacin was from Sigma Chemical Co; and beraprost sodium (sodium (±)-(1R*,2R*,3aS*,8bS*)-2,3,3a,8b tetrahydro-2-hydroxyl-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta-[b]benzofuran-5 butyrate) was supplied by Yamanouchi Pharmaceutical Co.

Isolation of Cardiac Fibroblasts
Cardiac fibroblasts were isolated by enzymatic extraction^{7 29} of left ventricles from inbred 4-month-old male La Jolla colony WKY and SHR rats. Ventricles were minced and digested at 37°C with 100 U/mL collagenase (Boehringer Mannheim) and 0.6 mg/mL pancreatin (Gibco BRL). After each of five successive digestions, isolated cells were centrifuged and resuspended in FBS. The collected cells were combined, pelleted, resuspended in DMEM containing 10% FBS and plated in 15-cm dishes for 35 minutes, after which nonadherent cells and debris were aspirated and fresh medium was added. This differential plating procedure yielded cultures of cells that were exclusively fibroblast at first passage.²⁹ Cultured rat fibroblasts were immunopositive for fibronectin, vimentin, and thy-1 and variably immunopositive for -smooth muscle actin, with SHR cells exhibiting somewhat greater frequency of positive reaction than WKY cells.⁷ Cells were negative for desmin and -sarcomeric actin and exhibited no labeling by DiI-Ac-LDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, acetylated low-density lipoprotein; Biomedical Technologies Inc), a marker of endothelial cell contamination. Bovine aortic endothelial cell cultures served as positive controls.

Measurement of AA Metabolite Release
Confluent cell cultures (10-cm plates) were used for metabolite release studies. DMEM with 0.5% FBS and [³H]AA (3 mCi/plate) were added, and cells were maintained at 37°C in 95% O₂/5% CO₂ for 18 hours to prelabel membrane phospholipids. The labeling medium was removed, adherent cells were rinsed with Ca²⁺/Mg²⁺-free PBS, pH 7.4, and 20 mmol/L HEPES/DMEM at 37°C was added for 30 minutes. Basal radiolabeled metabolite release during this "prestimulation phase" was estimated from 10% of the volume. For peptide-stimulated release, the desired concentration of peptide was added in a volume of incubation buffer equal to that removed, and the cells were incubated for the designated time. Total released radioactivity was determined in 10% of the incubation medium, and the balance was extracted with citric acid–acidified ethanol and ethyl acetate, as previously described.^{30 31} The extract was dried under nitrogen, redissolved, and subjected to HPLC analysis. Results were expressed as radioactivity (counts per minute) per milligram protein.³²

Measurement of PEt Formation
PLD activity was assayed as described by prelabeling cells with myristic acid and adding 400 mmol/L ethanol to form radiolabeled PEt.^{27 33} Confluent cultures (35-mm plates) were prelabeled for 24 hours in serum-free DMEM with [³H]myristic acid (2 mCi/plate) and BSA (1 mg/mL). Unincorporated radioisotope was removed by washing twice with 20 mmol/L HEPES, pH 7.4, 130 mmol/L NaCl, 5 mmol/L KCl, 1.5 mmol/L CaCl₂, 1 mmol/L MgCl₂, 0.1% (wt/vol) BSA, and 10 mmol/L glucose. After a 30-minute equilibration, 400 mmol/L ethanol was added and cells were incubated with either vehicle only (control) or vehicle with varying concentrations of Ang II or BK. Addition of 400 mmol/L ethanol did not affect cell viability as ascertained by both visual inspection and consistency in the cellular content of radioactivity per plate with or without added ethanol. Incubations were terminated by aspiring the medium and then adding 0.5 mL of ice-cold methanol/HCl (100:1, vol/vol). Cells were removed mechanically with an additional 0.3 mL of water and 0.5 mL of chloroform, and lipids were extracted.³⁴ Radioactive metabolites were identified by thin-layer chromatography on silica-gel plates (LK6DF, Whatman Inc) co-chromatographed with authentic PEt standard (5 mg, Avanti Polar Lipids) visualized by iodine staining. Chromatograms were developed with the upper phase from ethyl acetate/iso-octane/acetic acid/water (117:18:27:77, vol/vol). Segments (0.5 to 1.0 cm) of the silica gel were scraped, and the PEt fraction radioactivity was quantified. Results are expressed as the ratio (fractional percent) of radioactivity in the PEt segment relative to total radioactivity in the lane.

HPLC Separation of AA Metabolites
Gradient HPLC (Spectra-Physics Inc) resolved radiolabeled AA metabolites³⁵ at 20°C with a C₁₈ reversed-phase column (4.6x250 mm, Rainin Instrument Co). Development used a 40-minute linear gradient, 1.0 mL/min, from 50% Solvent A (water/acetic acid, 1:0.0008, pH 6.2, adjusted with ammonium hydroxide) and 50% Solvent B (methanol) to 100% solvent B. Radioactivity was determined in 0.4-mL (0.4 minute) fractions.

Measurement of Endogenous Production of PGI₂
Endogenous PGI₂ formed was assayed as 6-keto PGF₁, using an experimental design similar to that described above, but without added exogenous AA. Cell supernatants (35-mm plates) were centrifuged, and6-keto PGF₁ was assayed using an enzyme-linked immunoassay (Cayman Chemical). Results were expressed as nanogram per milligram protein.

Fibroblast Proliferation Analysis
Growth rates of fibroblast cultures (seeded at 10⁴ cells/cm² in 35-mm plates) were measured over a 4-day period either in control medium (vehicle) or with beraprost added (10 µmol/L). Cell number (triplicate cultures) was determined at 12-hour intervals by hemacytometer counting with Trypan blue counterstaining. DNA synthesis rate was measured by [³H]thymidine incorporation as described previously.³⁶ Subconfluent cultures (12-well dishes, 3.8 cm² ) were rendered quiescent by 24-hour serum deprivation, medium was removed, and 1 mL of treatment (in serum-free media) was added for 18 hours. Four hours before termination, [³H]thymidine (1 mCi/mL) was added. At 18 hours, the medium was aspirated, the cells were washed once with 0.5 mL of ice-cold PBS, twice with 0.5 mL of 10% (wt/vol) trichloroacetic acid, and once with 0.5 mL of methanol. The resulting precipitate was solubilized with sodium hydroxide (0.5 mol/L) and neutralized with hydrochloric acid, and radioactivity was measured.

RNA Extraction and Analysis
To measure steady-state mRNA levels for collagen types I and III, confluent cultures (15-cm dishes) were serum-deprived (24 hours) and incubated for 24 hours with either vehicle or beraprost (10 µmol/L). Total cellular RNA was extracted³⁷ and quantified by 260-nm absorbance, and Northern blot analysis was conducted.²⁹ Total RNA (5 or 10 µg/per lane) was separated on a 1% agarose gel containing 2.2 mol/L formaldehyde, transferred to a nylon membrane (Schleicher & Schuell), and probed with cDNA (supplied by Dr Noel Kim, University of California at San Diego) labeled with [-³²P]dCTP by random prime labeling (NEBlot, New England Biolabs). RNA was immobilized by ultraviolet light cross-linking and hybridized with the probes at 42°C for 16 hours. The membrane was washed at 55°C serially from 2x SSC/0.1% SDS to 0.2x SSC/0.1% SDS. For autoradiography, we used intensifying screens at -80°C. To correct for loading variations, filters were stripped and rehybridized with a 29-bp cDNA oligomer directed against 28S ribosomal RNA. All data are expressed as the ratio of mRNA/28S signal obtained from laser scanning densitometry.

Statistics
Results are reported as mean±SEM (replicate number in parentheses), and differences were assessed with ANOVA followed by Student's t test. The effects on cell proliferation were analyzed with two-way ANOVA and linear regression. Significance was accepted at P<.05.

	Results

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AA Metabolite Release
Total radioactive AA metabolite release from prelabeled cultures of WKY or SHR cardiac fibroblasts was monitored for 30 or 60 minutes after media change. The release rate was the same for both strains (WKY, k=0.051±0.01 min^-1; SHR, k=0.063±0.035 min^-1) and plateaued at 30 minutes after media change. Addition of 1 µmol/L peptide did not change the release rate, only the total amount of metabolites released. To compare the effect of Ang II and BK on fibroblasts from the different strains, analyses used a 30-minute stimulation period, and the effect was expressed as percent of vehicle control (Fig 1). With vehicle only, there was no difference in total AA metabolite release between SHR (4561±713 cpm/mg protein) and WKY (2994±955 cpm/mg protein) cultures. Addition of Ang II (1 µmol/L), but not BK (1 µmol/L), significantly increased AA metabolite release by WKY cells (P<.01). Neither peptide increased release from SHR cells relative to vehicle control.

View larger version (16K): [in this window] [in a new window]   Figure 1. [3H]AA metabolite release from cultured cardiac fibroblasts. Fibroblasts, prelabeled with [3H]AA, were stimulated for 30 minutes with vehicle, 1 µmol/L Ang II, or 1 µmol/L BK. Released metabolites were determined as described. Data are mean±SEM (n=4 to 5).

Measurement of PEt Formation
To examine strain differences in PLD activity and potential contribution to the release of endogenous AA, PLD activity was assayed using PEt formation and expressed as percent of vehicle control. There was no significant difference in PEt formation between SHR and WKY fibroblasts in vehicle only (SHR, 0.433±0.065% of radioactivity in PEt fraction; WKY, 0.427±0.046%). Ang II (1 µmol/L) tended to increase PEt formation in WKY (P<.08) but not SHR cardiac fibroblast cultures, whereas BK (1 µmol/L) did not increase PEt formation by cells of either strain.

HPLC Separation of AA Metabolites
Radiolabeled AA metabolites selectively released by Ang II or BK were analyzed by HPLC. A representative HPLC profile of WKY cells after Ang II stimulation (1 µmol/L) is shown in Fig 2. Peak identification relied on comparison with authentic standards and on the absence of the peak after preincubation of the cells with indomethacin (10 µmol/L), a cyclooxygenase inhibitor. The major metabolite resulting from Ang II stimulation of prelabeled cells comigrated with 6-keto PGF₁ standard, the stable metabolite of PGI₂, whereas the amount of PGE₂ and 12-HETE formed was variable.

View larger version (19K): [in this window] [in a new window]   Figure 2. Representative HPLC profile of [3H]AA metabolites released from WKY cardiac fibroblasts by Ang II (1 µmol/L) as described in "Methods."

Measurement of 6-Keto PGF₁
A specific enzyme-linked immunoassay was used to verify that cardiac fibroblasts produced authentic 6-keto PGF₁, as implied by HPLC, and to test whether cells produced PGI₂ from endogenous stores of AA in response to either Ang II (1 µmol/L) or BK (1 µmol/L). The results (Fig 3) were analyzed by two-way ANOVA, which identified a main effect of dose of Ang II (F=18.26; df=1,48; P<.0001) and a main effect of strain (F=10.39; df=5,48; P<.0001) but no interaction between peptide and strain. Analysis by Student's t test with Bonferroni correction indicated that the two strains differed only at the 1-µmol/L dose of Ang II. In the case of BK, there was a main effect of peptide (F=125.1; df=1,38; P<.0001), a main effect of strain (F=22.86; df=5,38; P<.0001), and an interaction between BK and strain (F=8.65; df=5,38; P<.0001). Analysis (Student's t test with Bonferroni correction) indicated that the two strains differed at the dosages shown (Fig 3).

View larger version (19K): [in this window] [in a new window]   Figure 3. Dose effect of Ang II and BK on endogenous 6-keto PGF1 release from cardiac fibroblasts measured by enzyme-linked immunoassay as described and expressed as ng/mg tissue protein content. Data are mean±SEM (n=5). *P<.05 vs control without Ang II or BK; #P<.05 SHR vs WKY.

Effect of Beraprost on Cell Proliferation
To assess potential consequences of PGI₂ release by cardiac fibroblast cultures, the effect of a stable PGI₂ analogue, beraprost sodium, on cell growth was examined and compared with control. Addition of beraprost (10 µmol/L) significantly (by two-way ANOVA and linear regression analysis) retarded cell growth rate of both SHR and WKY fibroblasts (WKY: F=5.60; df=7,48; P<.0001; SHR: F=3.91; df=7,48; P<.002); however, beraprost did not differentiate between the two strains (Fig 4). The effect of beraprost on growth was also evident (Fig 5) in the suppression of the rate of [³H]thymidine incorporation. There was a main effect of strain (F=1302; df=1,70; P<.0001) and of beraprost dose (F=48.71; df=4,70; P<.0001) and an interaction between strain and dose (F=12.69; df=4,70; P<.0001). For comparison, the lowest concentration of beraprost (10^-8 mol/L) decreased [³H]thymidine incorporation to 69% of control in WKY cells, whereas SHR cells required 10^-5 mol/L for a similar level of inhibition (62%).

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of 10 µmol/L beraprost on cell growth. Cardiac fibroblasts were treated with either vehicle or beraprost, and cell number was quantified as described. Solid symbol indicates vehicle-treated; open symbol indicates beraprost-treated. Data are mean±SEM of a single experiment (triplicate determinations).

View larger version (20K): [in this window] [in a new window]   Figure 5. Effect of beraprost on [3H]thymidine incorporation. Data are mean±SEM of two separate experiments with quadruplicate determinations. Significance of treatment was compared with control by one-way ANOVA.

Effect of Beraprost on Gene Expression for ECM Proteins
The effect of beraprost on collagen types I and III mRNA was analyzed by Northern blot analysis (Figs 6 and 7). Under basal conditions, both types of collagen mRNA were present in fibroblasts of the two strains; however, confirming previous reports,³⁸ SHR mRNA levels for both types of collagen were greater than WKY levels (P<.05). Beraprost reduced mRNA expression for both types of collagen in WKY cells (P<.05) but only for type III collagen mRNA in SHR cells There was a main effect of treatment for both type 1 (F=8.31; df=1,20; P<.01) and type III (F=170.09; df=1,20; P<.0001) and a main effect of strain (F=53.4; df=1,20; P<.0001; and F=815.7; df=1,20; P<.0001, respectively) but no interaction between treatment and strain (analyzed by two-way ANOVA).

View larger version (52K): [in this window] [in a new window]   Figure 6. Autoradiogram of Northern blot analysis of total RNA (10 µg per lane) from cardiac fibroblasts: vehicle-treated, lane 1; 10 µmol/L beraprost, lane 2. Membranes were probed first for collagen type I (coll-I), second for collagen type III (coll-III), and finally for 28S ribosomal RNA (28S).

View larger version (15K): [in this window] [in a new window]   Figure 7. Northern blot analysis of steady-state mRNA levels for vehicle- and beraprost-treated cultures. mRNA signals were quantitated by laser densitometry and corrected for differences in loading by the 28S ribosomal RNA. Data are mean±SEM (n=6). *P<.05 vs vehicle; #P<.05 SHR vs WKY.

	Discussion

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The results of the present study are the first to indicate that a primary eicosanoid formed by rat cardiac fibroblast cultures from endogenous AA is PGI₂, with only small and variable amounts of PGE₂ and 12-HETE found as assayed by HPLC. A negative test for incorporation of DiI-Ac-LDL argued against the possibility that the PGI₂ resulted from contaminating endothelial cells. The present result contrasts with an earlier report²⁶ in which PGE₂ was the primary eicosanoid formed by rat cardiac fibroblast cultures. The difference between the current and previous study likely lies with the experimental design, namely the source of the AA for metabolite formation. In the present study, measured AA metabolites were biosynthesized from cellular stores of AA, either after prelabeling cells for 18 hours with [³H]AA or from endogenous cellular sources of AA, whereas in the earlier report an excess of exogenous AA was added to the cells shortly before stimulation. Addition of excess AA at the time of stimulation, without permitting significant incorporation into membrane phospholipids, assays predominantly cyclooxygenase activity rather than terminal endoperoxide-metabolizing enzymes.^{30 39 40} Under such experimental conditions, PGE₂ would be the expected primary metabolite formed from PGG₂/PGH₂ and could arise by nonenzymatic routes.^{30 39 40 41} This difference in primary prostanoid product also mirrors an earlier disagreement in the primary prostanoid formed by 3T3L1 fibroblasts.^{42 43} When 3T3L1 microsomes were incubated with the endoperoxide percursor PGH₂, as substrate, the primary enzymatic product was PGI₂ with minimal PGE₂ formed,⁴² whereas the primary product released from 3T3L1 cells, after 1 hour of prelabeling the cells, and in response to the ionophore A23187, was PGE₂.⁴³ Again, the difference likely relates in part to experimental design. Short-term incubations with AA or overstimulation with A23187 would preferentially test cyclooxygenase activity rather than terminal endoperoxide-metabolizing activities. Our findings argue that PGI₂ would be the primary prostanoid product formed by rat cardiac fibroblasts from endogenous AA stores.

The present study also is the first evidence of strain differences in the formation of AA metabolites, particular PGI₂, by cardiac fibroblast cultures from WKY and SHR rats, and it extends previous findings^{7 8} of stable phenotypic differences between cultures of these strains. Although total AA metabolite release, under resting conditions, was the same between SHR and WKY cardiac fibroblasts, Ang II enhanced total metabolite release from WKY but not SHR cells, whereas neither cell type responded to BK. This effect was not due to the absence of Ang II receptors because we have documented receptors on cells from both strains.^{7 8} Release of radiolabeled AA from membrane phospholipids of prelabeled cells reflects an obligatory activation of a phospholipase, such as PLA₂, PLC, and/or PLD. Greater PLD activation by SHR vascular smooth muscle cells has been reported and linked to cell abnormalities.^{27 28} However, in the present study we found weak PLD activity in both SHR and WKY cardiac fibroblast cultures under resting conditions, and only WKY cultures indicated an enhanced activity after addition of Ang II, which did not reach significance. The pattern of PLD activation by Ang II paralleled that of AA metabolite release by the peptide. Because we previously found higher Ang II receptor density in WKY than in SHR cardiac fibroblasts,⁷ this higher density may explain strain differences in total metabolite release and PLD activation.

The use of both long-term prelabeling of cells with exogenous AA and endogenous phospholipid stores as sources for AA metabolites permits speculation on possible origins of strain-dependent differences. PGI₂ must result from the sequential actions of a phospholipase, cyclooxygenase, and PGI₂ synthase. WKY fibroblasts produced more PGI ₂ from endogenous AA than SHR cells, even under resting conditions. Because there were no strain differences in total AA metabolite release (or PLD activity) under resting nonstimulated conditions, we conclude that basal metabolism of intracellularly released AA to PGI₂ is greater in WKY than in SHR cells, which implies greater cyclooxygenase and/or PGI₂ synthase activity in WKY cells. After we added Ang II, WKY fibroblasts increased both total AA metabolite and 6-keto PGF₁ release, which suggests enhanced PGI₂ formation through phospholipase activation; this result is consistent with other studies^{44 45 46 47} because the Ang II AT₁ receptor subtype signals through activation of PLC and PLA₂. However, Ang II failed to enhance either total AA metabolite release or PLD activity by SHR cells, but it did increase 6-keto PGF₁ formation, as measured by enzyme-linked immunoassay. These strain-dependent differences argue that Ang II affects prostanoid synthesis by at least two mechanisms, one involving phospholipase activation and one distal to lipase activation; however, it is unclear from the present experiments whether the latter is at the cyclooxygenase or the PGI₂ synthase step.

From studies of BK activation of eicosanoid release by various cell types,^{14 31 47 48 49 50} it is clear that BK receptors activate both PLA₂ and PLC; however, in the present study BK neither enhanced AA metabolite release nor activated PLD in either cell strain, whereas it potently increased endogenous PGI₂ formation in WKY fibroblasts but only weakly in SHR cells. This dissociation between stimulated PGI₂ formation and release of total AA metabolites argues that BK, unlike Ang II, acts predominantly at a site distal to lipase activation; however, it cannot be concluded that the target sites affected by Ang II and BK are the same. Where might this target site be? Data in the literature are conflicting as to whether BK directly regulates cyclooxygenase activity. Based on results with Madin-Darby canine kidney cells, Coyne et al⁴⁸ argued that BK does not directly alter cyclooxygenase activity; however, Zhang et al³¹ reported that BK acted on vascular smooth muscle cells at the cyclooxygenase step in the AA cascade. Further studies are necessary to delineate whether enhanced PGI₂ formation by WKY fibroblasts reflects altered cyclooxygenase or PGI₂ synthase activity. Such a site may be the basis for the deficit in the production of PGI₂ by the SHR fibroblast under resting and stimulated conditions and may have implications in the control of ECM formation.

The present study also directly addresses the role of prostanoids in LV fibrotic development and remodeling and, indirectly, the contributions of Ang II and BK. The exact mechanism(s) by which ACEI modulate or reduce cardiac fibrosis and infarct size or improve cardiac performance and survival remains controversial, but a role for BK has been hypothesized.^{13 14 15 16 17 18} However, Gohlke et al¹⁷ have advanced the idea of an involvement of eicosanoids; they found that improvement of LV hypertrophy and cardiac function, such as LV pressure, differentiated LV pressure(dp/dt_max), or lactate production in stroke-prone SHR treated with ACEI correlated with increases in PGI₂ formation in the left ventricle. PGI₂ was also reported to directly relax coronary arteries as well as generate nitric oxide that mediates BK-induced vasodilation.⁵¹ The present study directly addresses this issue and establishes that PGI₂ is a major prostanoid product of BK action on rat cardiac fibroblasts and, in a companion study, on rabbit cardiac fibroblasts (A.M. Gallagher, H. Yu, M.P. Printz, unpublished data, 1997). One possibility is that ACEI-mediated enhanced formation of PGI₂ (resulting from stabilization of BK against degradation) contributes to the beneficial effects on the myocardium in an autocrine/paracrine manner. In support of this interpretation, we have shown that a PGI₂ analogue decreased the steady-state gene expression for collagen types I and III in both SHR and WKY cardiac fibroblast cultures and also affected cell proliferation. Early studies on lung fibroblasts implicated BK-mediated prostanoid formation (in this case PGE₂) with negative-feedback modulation of BK-enhanced protein and collagen formation,²² whereas others demonstrated that prostanoid enhancement of cAMP formation resulted in diminished collagen formation by diploid human foreskin fibroblasts.⁵² More recently, beraprost was shown to suppress proliferation of rat smooth muscle cells and inhibit their biosynthesis of glycosaminoglycans.²⁰ Transfection of PGI₂ synthase into rat smooth muscle cells increased PGI₂ synthesis and decreased DNA synthesis.⁵³ PGE₂ was shown to decrease human lung fibroblast proliferation²¹ and reduce collagen formation by both lung²¹ and cardiac fibroblasts²³ by inhibiting collagen synthesis and concomitantly enhancing its degradation. Because Brilla et al¹⁸ observed that lisinopril promoted collagen degradation via activation of matrix metalloproteinase I, the intervening mediator may also be a prostanoid. Based on the present findings and those of other investigators, we propose that locally generated prostanoids, particularly PGI₂, but possibly also PGE₂, modulate cardiac fibroblast activity, including collagen biosynthesis, and comprise one component of the cardioprotective effects of ACEI.

SHR cardiac fibroblasts have been shown to produce increased amounts of collagen, ie, increased expression of mRNA for collagen types I and III compared with WKY cells, and this difference in expression was proposed to potentially contribute to LV hypertrophy in genetic hypertension.³⁸ In the present study, we also found increased mRNA levels for collagen types I and III in SHR compared with WKY cultures, which compares favorably with the report of increased collagen types I and III mRNA in vivo in the left ventricle of SHR compared with WKY.⁵⁴ Because the present study has shown that SHR cardiac fibroblasts are less responsive than WKY cells to BK-mediated PGI₂ formation and less responsive to PGI₂ inhibition of collagen mRNA formation, this implies that the SHR myocardium would be at greater risk than WKY to fibrotic development after acute injury or with sustained pressure overload. Such a conclusion can be experimentally tested.

In conclusion, we have found that a primary prostanoid formed by rat cardiac fibroblasts is PGI₂ and that this eicosanoid exerts inhibitory actions on fibroblast proliferation, DNA synthesis, and steady-state mRNA formation of collagen types I and III. We also have demonstrated that differences in the ability to form PGI₂ and in the cellular response to PGI₂ constitute yet another phenotypic difference between cultures of cardiac fibroblasts from SHR and WKY, and this difference may be relevant to the target organ damage to the heart in genetic hypertension.

	Selected Abbreviations and Acronyms

 

AA = arachidonic acid ACEI = angiotensin-converting enzyme inhibitors Ang II = angiotensin II BK = bradykinin DMEM = Dulbecco's modified Eagle's medium ECM = extracellular matrix FBS = fetal bovine serum HPLC = high-pressure liquid chromatography LV = left ventricular PEt = phosphatidylethanol PGE2 = prostaglandin E2 PGF1 = prostaglandin F1 PGI2 = prostaglandin I2 PLA2 = phospholipase A2 PLC = phospholipase C PLD = phospholipase D SHR = spontaneously hypertensive rats

	Acknowledgments

 
This work was supported in part by grants from the Japanese Gerontology Foundation (H.Y.) and by National Institutes of Health, National Heart, Lung, and Blood Institute grant HL-35018 (M.P.P.).

Received December 2, 1996; first decision January 2, 1997; accepted April 23, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 

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